Small Evolutionarily Conserved RNA, Resembling C/D Box Small Nucleolar RNA, Is Transcribed from PWCR1, a Novel Imprinted Gene in the Prader-Willi Deletion Region, Which Is Highly Expressed in Brain  Tala de los Santos, Johannes Schweizer, Christian A. Rees, Uta Francke  The American Journal of Human Genetics  Volume 67, Issue 5, Pages 1067-1082 (November 2000) DOI: 10.1086/303106 Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 1 a, Monoallelic expression of PWCR1, demonstrated by RT-PCR analyses of control (lanes N1 and N2), AS (lanes A1 and A2), and PWS (lanes P1 and P2) lymphoblastoid cell lines. Primers H1TD and H2TD amplified a 690-bp product (arrowhead) from control and AS cell lines but not from PWS cell lines. β-Actin–specific primers were included as positive controls. A plus sign (+) denotes presence of reverse transcriptase; a minus sign (−) denotes its absence. Lane N1, LCL 1497. Lane N2, LCL 1385. Lane A1, LCL 1201. Lane A2, LCL 1101. Lane P1, LCL 863. Lane P2, LCL 1309. b, Schematic map of known imprinted genes in the human 15q11.2 region. PWCR1 is located centromeric to IPW. PWCR1 probes hybridize to PAC pDJ134I14, PAC pDJ121D5, cosmid 88, and cosmid 72. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 2 a, Sequence of PWCR1 cDNA clone. The 5′ and 3′ repeats (called “HMCR” [human-mouse conserved region]) are boxed. The imperfect putative poly-A signal is in boldface. b, Probes used for analysis of PWCR1 expression, by northern blot. Probes hA and hB are double-stranded DNA probes. Probes hB-F and hB-R are PCR-labeled single-stranded DNA probes. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 3 Northern blot analyses of polyadenylated RNA, with probes indicated. a, Human adult tissues. b, Different regions of human brain. c, Human fetal tissues. Arrowheads indicate major PWCR1 transcripts. d, Direction of transcription of PWCR1. Identical northern blots contain total RNA from a normal control brain. Both the 1.2-kb and the 140-nt transcripts were detected only with probe hB-R. β-actin control probes were used to assess the amount of RNA loaded. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 4 Expression of PWCR1 transcripts exclusively from the paternal allele. Northern blots were hybridized with either probe hA or probe hB, as indicated. a, Total lymphoblast RNA from control individuals (lane N1) and from individuals with either AS (lanes A1 and A2) or PWS (lanes P1 and P2). b, Total brain RNA from a control (lanes N) and a PWS subject (lanes PWS). A β-actin probe was used as control for RNA loading. Arrowheads indicate PWCR1 transcripts. c, RT-PCR using PWCR1-specific primers H58JS and H59JS on brain RNA from a control individual (lanes N), which amplified a 548-bp product. No PWCR1-specific product was observed in the PWS brain RNA sample, whereas β-actin–specific primers amplified products in both samples. A plus sign (+) denotes the presence of reverse transcriptase; a minus sign (−) denotes its absence. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 5 a, Simplified map of the region surrounding PWCR1, showing both the location of PWCR1-like truncated copies (PWCR1L1–PWCR1L25) and the direction of transcription (arrows). Hatched boxes within x102 represent sequences similar to PWCR1. The arrow below x102 indicates the proposed direction of transcription, according to GenBank annotation (accession number AF017338). b, Dendrogram of relationships between PWCR1 and the various truncated copies, based on sequence similarity (also see table 1). The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 6 a, Comparison of human (H) and mouse (M) genes, which reveals that PWCR1 and Pwcr1 cDNAs contain a block of conserved sequences (HMCR). Arrows indicate direction of transcription. Probes mC, mD, and mE are double-stranded DNA probes. Probes mD-F and mD-R are RNA probes. b, Alignment of human (H) and mouse (M) HMCRs. Sequences are 87% identical over 99 nucleotides. Conserved sequences representing C, D′, and D boxes that identify a class of snoRNAs are boxed (Smith and Steitz 1998). c, Sequence of Pwcr1 derived from cDNA clone and HTGS data. The HMCR and the AC-repeat region are boxed. The sequence identical to Ipw exon A1 (nt 644–789) is in italics. d, Mapping of Pwcr1 to mouse chromosome 7, in a conserved syntenic region with human 15q11.2. The Jackson Laboratory Mapping Panels BSS backcross was typed for a Pwcr1 fragment-length polymorphism. Raw typing data for other markers in the region also were obtained from The Jackson Laboratory Mapping Panels Web site. Missing typing was inferred from surrounding data, wherever assignment was unambiguous. Top, BSS backcross map of chromosome 7. The centromere is at the top of the map. Loci mapping to the same position are listed in alphabetical order. Bottom, Haplotypes from the BSS backcross. Loci are listed in order, with the most proximal at the top. Black boxes represent the C57BL6/JEi allele, and white boxes represent the SPRET/Ei allele. Listed at the bottom of each column is the number of animals with each haplotype. R = percent recombination for adjacent loci. SE = standard error for each R. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 7 Local cluster of Pwcr1-like repeat units. a, Organization of head-to-tail copies within the GenBank sequence (accession number AC026683). b, Dendrogram of relationships between Pwcr1 and the Pwcr1-like genes, based on sequence similarity, which suggests expansion of the cluster by local duplication events. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 8 Tissue-specific and imprinted expression of Pwcr1. Northern blot analyses of polyadenylated mRNA from adult mouse tissues (a) and total mouse embryos (b). Arrowheads indicate Pwcr1 transcripts. dpc = days postcoitum. c, Pwcr1 expressed exclusively from the paternal allele. RT-PCR was performed with Pwcr1-specific primers M49TD and M50TD, on RNA from mutant mice deleted for exons 1–6 of Snrpn and for the putative imprinting center (IC) region (lanes C1 and lanes IC2) and from their wild-type littermates (lanes WT1 and lanes WT2). A 208-bp product was amplified in WT1 and WT2 but not in either IC1 or IC2. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions

Figure 9 Southern blot analyses of various animal species, which reveal conservation of PWCR1-related sequences in most mammals. Mouse probe mD that contains the HMCR was used (see fig. 6a). DNA samples from dog, pig, baboon, and human were digested with BamHI; the other DNA samples were digested with EcoRI. The American Journal of Human Genetics 2000 67, 1067-1082DOI: (10.1086/303106) Copyright © 2000 The American Society of Human Genetics Terms and Conditions